This study was aimed at exploring the effects of miR-215 and its target gene stearoyl-CoA desaturase (SCD) on colorectal cancer (CRC) cell migration and invasion.  Here, we analyzed the relationship between miR-215 and SCD, as well as the regulation of miR-215 on CRC cells. We constructed wild-type and mutant plasmids of SCD to identify whether SCD was a target gene of miR-215 by using a luciferase reporter assay. The expression of miR-215 and SCD was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. MTT, wound healing, and Transwell assays were applied to determine the effect of miR-215 on CRC cell proliferation, migration, and invasion. in vitro. In addition, the result of rescue experiments showed that overexpression of SCD could promote the proliferation, migration, and invasion of CRC cells, and the carcinogenic effect of SCD could be inhibited by miR-215.  It was found that miR-215 expression was significantly decreased in CRC tissue while SCD was highly expressed compared with those in adjacent normal tissue. The luciferase reporter assay indicated that SCD was a direct target gene of miR-215. Functional analysis revealed that miR-215 overexpression significantly inhibited CRC cell proliferation, migration, and invasion  Taken together, our findings suggested that miR-215 could inhibit CRC cell migration and invasion via targeting SCD. The result could eventually contribute to the treatment for CRC. Colorectal cancer (CRC) is one of the common malignant cancers of the digestive tract, including the rectum and colon, which mainly occurs in the rectum and the junction between the rectum and sigmoid colon. CRC is also the third most frequently diagnosed cancer and the fourth leading cause of cancer-related death globally , 2. Althβ-catenin signaling pathway [Stearoyl-CoA desaturase (SCD) is a key enzyme for the formation of monounsaturated fatty acids from saturated fatty acids, and its main components include palmitoleic acid (C16:1) and oleic acid (C18:1) . In rece pathway . SCD can pathway . HoweverMicroRNAs (miRNAs) are a class of small noncoding RNAs that can regulate gene expression by facilitating mRNA degradation or inducing translational repression . The miRIn this study, we tested miR-215 expression in CRC cells and investigated the biological effect of miR-215 on migration and invasion of CRC cells. Here, we found that miR-215 exerted a suppressive effect on tumor migration and invasion by targeting SCD.https://www.ncbi.nlm.nih.gov/geo/) with “Colorectal cancer” as the key word, and two miRNA microarrays GSE110224 and GSE35834 were selected. GSE110224 included 17 normal samples and 17 tumor samples with histologically confirmed CRC. Total mRNAs were extracted for further processing with the Human Genome U133 Plus 2.0 Array . GSE35834 contained 78 samples comprising 23 normal adjacent mucosa tissue samples and 55 CRC tumor samples, and GPL8786 Multispecies miRNA-1 Array  was used as the sequencing platform. The datasets were analyzed with the R package “Limma.” ∣logFC | >1 and p value < 0.05 were set as the threshold for screening the differentially expressed genes (DEGs).We searched the Gene Expression Omnibus (GEO) database (http://starbase.sysu.edu.cn/), TargetScan (http://www.targetscan.org/), and miRTarBase (http://mirtarbase.mbc.nctu.edu.tw/) databases. A Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn/) was used to find the intersections of the predicted results in the three databases.The miRNAs that regulate SCD were retrieved in the starBase V2.0  were seeded in a six-well plate and then subjected to transfection by employing Lipofectamine 2000 .The CRC cell line HT29 was obtained from the Bena Culture Collection  and was grown in Dulbecco's Modified Eagle's Medium  with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% fetal bovine serum (FBS). All cells were maintained in a humidified incubator with 5% CONC (transfected with negative sequence), miR-215 mimic, and miR-215 inhibitor were purchased from GeneCopoeia . SCD overexpression (oe-SCD) and corresponding negative control (oe-NC) were constructed by lentiviral vectors.Target sequences of wild-type (WT) and mutant (WUT) SCD 3′UTR were constructed artificially and ligated into the pmirGLO  reporter plasmids with enzymes BamHI and XhoIII to obtain WT and MUT reporter plasmids. Afterwards, the two reporter plasmids were cotransfected with the miR-215 mimic or NC into the cancer cell line using Lipofectamine 2000. Relative luciferase activities were determined by the Dual-Luciferase® Reporter Assay System (Promega) following the instructions 48 h after transfection.ΔΔCT- method with U6 and GAPDH as the internal reference for miR-215 and SCD, respectively. miR-215 stem-loop primers were as follows: 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGTCTGT-3′. The sequences of the PCR primers were as follows: miR-215 forward 5′-CTCAACTGGTGTCGTGGAGTCGG-3′ and reverse 5′-ACAGGAAAATGACCTATGAATTGAC-3′, U6 forward 5′-GTACAAAATACGTGACGTAGAAAG-3 and reverse 5′-GGTGTTTCGTCCTTTCCAC-3′, SCD forward 5′-TCTAGCTCCTATACCACCACCA-3′ and reverse 5′-TCGTCTCCAACTTATCTCCTCC-3′, and GADPH forward 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′.Total RNA was extracted from CRC cells, tumor tissue, and paired adjacent normal tissue using Trizol Reagent  according to the manufacturer's instructions. The concentration and purity of RNA were determined with an ultraviolet spectrophotometer. RNA was reversely transcribed into cDNA by using RT-PCR Kit , and quantitative real-time- (qRT-) PCR was performed according to the manufacturer's instructions of SYBR Premix Ex Taq II (TaKaRa). The relative expression level of RNA was calculated by the 2Cells in the logarithmic growth phase were collected and lysed with the RIPA buffer containing protease and phosphatase inhibitors. Extracted proteins were loaded onto 8% SDS-PAGE at a voltage of 150 V after quantification. Thereafter, the proteins were transferred onto the PVDF membrane and incubated with the primary mouse antibodies against SCD  and GADPH , respectively, overnight at 4°C. After three times being washed with TBST, the membrane was probed with the secondary antibody rabbit anti-mouse IgG (HRP)  for 1 h at room temperature. Finally, the protein bands were detected using the ABI 7500 Real-Time PCR System .5 cells/mL. Then, the cells were seeded in 96-well plates at a density of 2 × 103 cells/well with the volume of 200 μL. 5 mg/mL of MTT solution was added to each well at 24 h, 48 h, and 72 h, respectively. After incubation for 4 h, the reaction was stopped. The supernatant was discarded after centrifugation, and 100 μL of DMSO was added to each well to promote crystal dissolution. The absorbance was measured at 490 nm. The assay was performed in triplicate.The MTT assay was conducted to evaluate cell proliferation capacity. Cells were trypsinized after 24 h of culture in serum-free medium. The cells were counted with a hemocytometer, and the cell density was adjusted to 1 × 105 cells/well) with marks on the back of the plate. After 24 h of culture, the cells covered the entire plate, and scratches were created perpendicular to the marks using a 10 μL pipette. The detached cells were washed away with PBS, followed by the addition of serum-free DMEM. Cells were incubated in an incubator with 5% CO2 at 37°C, and images at 0 h and 48 h were captured under an inverted microscope. Three parallel wells were prepared for each group.CRC cells in the logarithmic phase were inoculated into a 6-well plate  covered with Matrigel were used for a cell invasion assay. CRC cells in the logarithmic phase were harvested for digestion with trypsin, then washed once with PBS and resuspended in serum-free DMEM. The cell density was adjusted to 1 × 10t-test, while comparisons among multiple groups were assessed by one-way ANOVA. Counting data were analyzed by the chi-square test. P < 0.05 was considered statistically significant.Statistical analysis was conducted using SPSS 21.0 software and GraphPad Prism 6.0. Experiment data were recorded as the mean ± standard deviation. Comparisons between two groups were assessed by Student's The transcriptome expression data of CRC were analyzed by the bioinformatics method. The results showed that SCD was significantly upregulated in CRC samples compared with normal samples Figures . At the In order to explore the underlying mechanism of SCD in CRC cells, we firstly analyzed the GSE35834 dataset and obtained 20 miRNAs with significant differential expression in CRC . Then, tTo confirm whether SCD was a direct target gene of miR-215 in CRC cells, we conducted the luciferase reporter assay in HT29 cells 48 h after cotransfection of WT or MUT SCD and miR-215 mimic or NC. The result exhibited that overexpression of miR-215 reduced the luciferase activity of WT SCD in HT29 cells but had no effect on MUT SCD . AdditioAccording to the aberrant expression of miR-215 in CRC cells, we speculated that it might regulate cancer cell proliferation, invasion, and migration. In order to test our hypothesis, we overexpressed and inhibited miR-215 in HT29 cells by transfecting the miR-215 mimic and inhibitor, respectively. qRT-PCR was used to confirm that the miR-215 expression was decreased significantly in the miR-215 inhibitor group and increased in the miR-215 mimic group . After tIn order to verify that miR-215 can regulate the cellular function of CRC cells by inhibiting the expression of SCD, we conducted the rescue experiments. Firstly, we detected the mRNA and protein levels of SCD in three transfected HT29 cell lines , and the results showed that the elevated expression of SCD was downregulated by miR-215 Figures . Then, wMore than 1.2 million patients are diagnosed with CRC each year. The mortality of CRC is the fourth highest of all the cancer deaths, and the disease tends to be younger in epidemiology. Therefore, studying the mechanism of occurrence, invasion, and migration of CRC can improve the therapeutic effect of CRC patients. Accumulating evidence shows that miRNAs are closely related to the proliferation, invasion, migration, and recurrence of various tumors, and they can be used as effective molecular markers as well as therapeutic targets for cancer diagnosis, prognosis, and treatment –15.In this study, we found that SCD was highly expressed in CRC by bioinformatics. The literature on SCD has also showed that SCD is upregulated in multiple cancers, such as ovarian cancer , breast in vitro by inhibiting the expression of SCD. The results of this study contribute to the improvement of the understanding of the molecular mechanism underlying CRC development and progression and provide potential new therapeutic targets for the management of CRC.In summary, we demonstrated that the expression of miR-215 was downregulated in CRC tissue compared with adjacent normal colorectal tissue. miR-215 impaired CRC cell proliferation, migration, and invasion


We developed a new format for liquid crystal (LC)-based multi-microfluidic immunoassays, hosted on a polydimethylsiloxane substrate. In this design, the orientations of the LCs were strongly affected by the interface between the four microchannel walls and surrounding LCs. When the alignment layer was coated inside a microchannel, the LCs oriented homeotropically and appeared dark under crossed polarizers. After antigens bound to the immobilized antibodies on the alignment layer were coated onto the channel walls, the light intensity of the LC molecules changed from dark to bright because of disruption of the LCs. By employing pressure-driven flow, binding of the antigen/antibody could be detected by optical signals in a sequential order. The multi-microfluidic LC biosensor was tested by detecting bovine serum albumin (BSA) and an immunocomplex of BSA antigen/antibody pairs, a protein standard commonly used in labs. We show that this multi-microfluidic immunoassay was able to detect BSA and antigen/antibody BSA pairs with a naked-eye detection limitation of −0.01 µg/mL. Based on this new immunoassay design, a simple and robust device for LC-based label-free microfluidic immunodetection was demonstrated. Microfluidic immunoassays based on polydimethylsiloxane (PDMS) substrates possess several advantages such as short assay times, small volumes, and low costs ,2. HowevRecently, liquid crystals (LCs) were successfully used for label-free biodetection . It was Based on the problems mentioned above, we planned to take advantage of the fluidic property of LC materials, as well as propose an LC-based multi-microfluidic immunoassay to detect BSA antigen/antibody pairs. However, some efforts were proposed for integrating LC materials into microfluidic devices. The first LC/PDMS film to detect ethanol in microfluidic devices was recently proposed . The devIn this study, we developed an LC-based multi-microfluidic immunoassay chip. We show the behavior between BSA antigen/antibody pairs and LC molecules in a PDMS microchannel. We show that this device substantially differs from a typical optical cell. Since the LC molecules within a microchannel are in contact with the four alignment-coated channel walls, the orientations of the LCs are determined by both the surface functionality and the geometric dimensions of the channels. Antigens/antibodies can be detected by observing the optical appearance of LCs inside the microchannel under crossed polarizers. A highly sensitive interface between LC molecules and an alignment layer of N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP) was employed to detect BSA concentrations. The schematic of this multi-microfluidic LC/PDMS biosensor is shown in Single-layer and two-level cascaded microchannels were fabricated by a general PDMS-based soft lithographic fabrication process. First, a microchannel mold was fabricated on a 4-inch (10.16-cm) silicon wafer by standard photolithography using SU-8 2025 photoresist. The thickness of the mold was 25 μm. Then the PDMS base and curing agent were mixed in a 10:1 weight ratio. After allowing 30 min for degassing, the mixture was poured into the master. After baking at 65 °C for 4 h, the cured PDMS microchannel was peeled off the master. Using oxygen plasma treatment, the PDMS microchannel and a pre-cleaned glass substrate were tightly bonded together. The LC mixture used in this study was nematic E44. In addition, the multi-microfluidic channels were immersed in the DMOAP aqueous solution for 30 min to coat the aligned layer onto the inner walls of the microfluidic channel. Next, the multi-microfluidic device was rinsed with deionized water for 1 min in order to remove excess DMOAP solution. For test immobilization, BSA in the form of an aqueous solution was filled into the DMOAP-coated microfluidic channel. In addition, BSA concentrations of 1 and 0.1 mg/mL, and 10, 1, 0.1, and 0.01 µg/mL were used. Concentrations of an anti-BSA antibody of 0, 1, 10, and 100 µg/mL were also used. The empty microfluidic channel was filled with LC under volume flow rates of 5, 10, 20, and 30 µL/min through a syringe to form LC multi-microfluidic chips. A BX51 polarized optical microscope  equipped with a halogen light bulb as the light source was used in this study for observation. All experimental data were acquired at 26 ± 1 °C.y) and light intensity (x) was y = 0.49x. These experimental results proved that the LCs in the multi-microfluidic can also be used for quantitating and detecting biomolecules in a linear manner as a new label-free biosensor.Polarized optical images of the LC multi-microfluidic biosensors with BSA concentrations of 0 and 1 mg/mL under crossed and paralleled polarizers are shown in In addition, an immunoassay test of the device was also examined using both BSA and an anti-BSA antibody. Intensities of the immunoassay LC microfluidic devices immobilized with 0, 1, and 10 μg/mL concentrations of BSA and 0, 10, 100, and 1000 µg/mL concentrations of the anti-BSA antibody are shown in Volume flow rates in LC microfluidic devices are important because of the fluidity of LC molecules. Moreover, many label-free biosensing techniques were proposed in the past. The most important and commonly used techniques are grating coupled interferometry (GCI) and plasmonic sensing ,31,32,33A novel design of LC-based multi-microfluidic immunoassay chips was proposed. The orientation of the LCs was strongly influenced by the interface between the four channel walls and surrounding LC molecules. When a DMOAP alignment layer was coated onto the interior of the microchannel, the LCs oriented homeotropically and appeared dark under crossed polarizers. After the antigens had bound to the immobilized antibodies in the multi-microfluidic device, the appearance of the LC phase changed from dark to bright because of disruption of the LC orientation. Using pressure-driven flow, antigen/antibody binding could be detected by optical signals in a sequential order. The immunodetection limit of BSA antigen/antibody pairs was 0.01 µg/mL BSA and 1 µg/mL of the anti-BSA antibody. We show that this multi-microfluidic LC immunoassay chip is able to detect BSA and antigen/antibody BSA immunocomplexes with label-free immunodetection. This new design of an immunoassay device provides a sensitive, cheap, multi-detection, and robust approach to LC-based immunodetection.


The aim of this study was to develop and validate a scale to measure nursing students’ readiness for the flipped classroom in Sri Lanka.A literature review provided the theoretical framework for developing the Nursing Students’ Readiness for Flipped Classroom (NSR-FC) questionnaire. Five content experts evaluated the NSR-FC, and content validity indices (CVI) were calculated. Cross-sectional surveys among 355 undergraduate nursing students from 3 state universities in Sri Lanka were carried out to assess the psychometric properties of the NSR-FC. Principal component analysis , internal consistency , and confirmatory factor analysis  were done to test construct validity and reliability.2/degrees of freedom=1.57).Thirty-seven items were included in the NSR-FC for content validation, resulting in an average scale CVI of 0.94. Two items received item level CVI of less than 0.78. The factor structures of the 35 items were explored through PCA with orthogonal factor rotation, culminating in the identification of 5 factors. These factors were classified as technological readiness, environmental readiness, personal readiness, pedagogical readiness, and interpersonal readiness. The NSR-FC also showed an overall acceptable level of internal consistency (Cronbach α=0.9). CFA verified a 4-factor model  and 20 items that achieved acceptable fit (standardized root mean square residual=0.08, root mean square error of approximation=0.08, comparative fit index=0.87, and χThe NSR-FC, as a 4-factor model, is an acceptable measurement scale for assessing nursing students’ readiness for the flipped classroom in terms of its construct validity and reliability. Contemporary nursing education aims to prioritize student-centred learning, which is perceived as high-order, flexible, and individualized . Recent The FC has entered into use in undergraduate nursing education and has been in the spotlight in discussions of the implementation of nursing curricula ,3. With The aim of this study was to develop and validate a tool to measure nursing students’ readiness for the FC, namely the Nursing Students’ Readiness for Flipped Classroom (NSR-FC). Content validity, construct validity, and reliability tests were done to validate the measurement scale.The study was approved by the University of Auckland Human Participants Ethics Committee . Participants were provided with an information sheet before the anonymized questionnaire was administered. Participants were clearly informed that the voluntary return of the questionnaire to the collection box indicated their consent to participate in the anonymized survey.This was a psychometric study to validate the measurement scale based on experts’ opinion and survey results for the scale.Five experts participated in validity testing of the scale, and 265 undergraduate nursing students (in the 1st and 2nd academic years) from 3 state universities  in Sri Lanka participated in exploratory factor analysis (EFA). Responses from 90 different undergraduate nursing students (3rd and 4th academic years)  were employed for confirmatory factor analysis (CFA).This cross-sectional study was conducted in 2 steps. The first step involved the development of the NSR-FC. The second step was to investigate the psychometric properties of the NSR-FC.The NSR-FC was drafted after reviewing the literature pertaining to learner readiness. The following existing inventories were used to generate items for the NSR-FC: E-Learning Readiness ; Online Content validation was done to assess the level of representativeness, relevance, understandability, and completeness of the NSR-FC. Five Sri Lankan content experts including 3 senior academics in health profession education and 2 academics in nursing participated and individually evaluated the degree of item significance for nursing students’ readiness to engage in the FC. The content validity indices (CVI) at the average scale-level (S-CVI/average) and item-level (I-CVI) were calculated using descriptive statistics. The I-CVI for each item on the NSR-FC was computed as the number of experts giving a rating of 1 (not relevant) to 4 (highly relevant) divided by the total number of experts who responded to the item. An I-CVI score of 0.78 or higher was considered to be adequate . The S-CEFA and CFA were conducted for the investigation of construct validity of the NSR-FC. In total, 265 undergraduate nursing students participated in EFA. The Kaiser–Meyer–Olkin (KMO) test and the Bartlett test of sphericity were used to test the adequacy of the study’s sample and the suitability of orthogonal factor rotation . Princip2/degrees of freedom [df], with a threshold of <5) [The degree of model fit was assessed through CFA, which utilized responses from 90 different undergraduate nursing students (Dataset 2). Four goodness-of-fit indices were calculated to estimate the global fit of the NSR-FC: the comparative fit index ; the root mean square error of approximation ; standardized root mean square residual ; and the chi-square statistic and its ratio to degrees of freedom (χd of <5) ,13.Internal consistency is commonly used to indicate the degree of reliability of a self-reporting questionnaire. The internal consistency of the NSR-FC was measured by computing the Cronbach α coefficient. A Cronbach α of 0.70 or higher was considered to indicate acceptable internal consistency, meaning that the observed score variance is reliable when compared with the true score variance .In accordance with the recommendation made by Lynn , 5 conteDescriptive statistics were employed to calculate CVI measures using Microsoft Excel . PCA was applied using IBM SPSS ver. 26.0  to explore the factor structure. Cronbach α coefficients were computed using IBM SPSS ver. 26.0 (IBM Corp.). The CFA goodness-of-fit indices were calculated using AMOS ver. 26.0 (IBM Corp.) ,10.At the item level, 91.9% of the NSR-FC’s items (n=34) had an I-CVI greater than or equal to 0.90. Two items of the NSR-FC (E1 & E2) received an I-CVI less than 0.78, namely “I can discipline myself to follow flipped learning” (I-CVI=0.75) and “I am committed to using flipped learning” (I-CVI=0.45). In addition, reviewers reported that E1 and E2 duplicated existing items, so these 2 items were excluded from the questionnaire. The S-CVI/average of the questionnaire achieved an acceptable level of 0.94 .2=4,717.18, P<0.001). The PCA revealed that NSR-FC could be reduced to 5 factors, namely technological readiness, environmental readiness, personal readiness, pedagogical readiness, and interpersonal readiness. Parallel analysis confirmed the 5 factors in the NSR-FC. The percentage of variance explained by the rotated factor matrices ranged from 4.29% to 25.42% per factor, with the 5 factors explaining 55.25% of the overall variance. Factor loading after the rotation of each item is shown in  included 12 items (Q6–16 and Q18). The factor had a cumulative eigenvalue of 8.89 and accounted for 25.42% of the total variance. Environmental readiness (factor 2) comprised 5 items (Q20–21 and Q23–25), had a cumulative eigenvalue of 4.38, and accounted for 12.51% of the total variance. Personal readiness (factor 3) contained 6 items . The factor had a cumulative eigenvalue of 2.81, and accounted for 8.02% of the total variance. Pedagogical readiness (factor 4) encompassed 7 items , had a cumulative eigenvalue of 1.75, and accounted for 5.01% of the total variance. Interpersonal readiness (factor 5) included 4 items (Q30–33) and had a cumulative eigenvalue of 1.50, accounting for 4.29% of the total variance. Item Q5 obtained a factor extraction value of 0.25, which was deemed to be low; this item was therefore excluded from the NSR-FC.The Cronbach α values of the NSR-FC’s factors ranged from 0.76 to 0.93, and the Cronbach α for the scale as a whole was 0.90, indicating excellent reliability. The intraclass correlation coefficients (ICCs) for the NSR-FC’s factors showed satisfactory results for each subscale , and the overall ICC was 0.90, exhibiting an acceptable level of reliability. The corresponding data are presented in .2/df. Therefore, model 2 was created by excluding 5 items  that showed loading values less than 0.4; however, the goodness-of-fit values of model 2 were still below the cut-off level , model 3 achieved an acceptable level of fit (SRMR=0.08 and χ2/df=1.57). In reference to RMSEA, model 3 demonstrated reasonable fit (RMSEA=0.08). The CFI of model 3 (0.87) came close to meeting the cut-off level (In addition, all items in factor 5 did not obtain an acceptable loading value. Therefore, it was decided to exclude factor 5 from model 3. Model 3 was found to be the model with the best fit in the current study . Accordiff level .Student readiness is recognized as a valuable factor for determining pedagogical effectiveness . TherefoThe method used to construct the NSR-FC was similar to the procedure used for the development of student readiness scales in other disciplines, and included proposing constructs, item generation, analysis of the content, item reduction, and validation of the newly developed instrument ,7. PCA rThe 4 best-fitting factors of the NSR-FC were comparable with existing inventories used to measure students’ readiness for blended learning in school-level education. Technological readiness is a common subset, identified as technology self-efficacy in many other studies ,18. TechIn the reliability assessment, the NSR-FC demonstrated acceptable internal consistency. All 4 factors of the NSR-FC generated Cronbach α and ICC values greater than 0.7, confirming internal consistency within the domains and the ability of the NSR-FC to generate reproducible results. Therefore, the 4-factor model of NSR-FC is a valid and reliable tool that can be used to measure students’ readiness.This instrument can be used by nursing educators and curriculum planners to evaluate the effectiveness of FC pedagogy. However, the results of factor analysis may have been sample-specific, and the generalizability of these results is subject to the similarity of respondents to the sample. Since the study was conducted in Sri Lanka, the NSR-FC could be used to assess nursing students’ readiness for the FC in the context of South Asia. Moreover, the CFI value of the NSR-FC did not meet the acceptable level. This may have been due to the limited sample size and is acknowledged as a limitation of the study. Thus, future research is encouraged to perform CFA of the NSR-FC with a larger sample size. Lastly, the study was conducted through a self-reported questionnaire survey, which may have resulted in reporting biases such as social desirability bias . QualitaThese findings indicate that the NSR-FC is an acceptable instrument for measuring nursing students’ readiness for the FC in reference to its construct validity and reliability within the Sri Lankan nursing education context. The 4 readiness subscales were found to be technological readiness, environmental readiness, personal readiness, and pedagogical readiness. This finding may provide a good platform and frame of reference for nursing curriculum researchers and educational designers regarding the necessity of assessing students’ readiness for gaining actual educational achievements through the FC.


The structural and electronic properties of interfaces composed of donor and acceptor molecules play important roles in the development of organic opto-electronic devices. Epitaxial growth of organic semiconductor molecules offers a possibility to control the interfacial structures and to explore precise properties at the intermolecular contacts. 5,6,11,12-tetraazanaphthacene (TANC) is an acceptor molecule with a molecular structure similar to that of pentacene, a representative donor material, and thus, good compatibility with pentacene is expected. In this study, the physicochemical properties of the molecular interface between TANC and pentacene single crystal (PnSC) substrates were analyzed by atomic force microscopy, grazing-incidence X-ray diffraction (GIXD), and photoelectron spectroscopy. GIXD revealed that TANC molecules assemble into epitaxial overlayers of the  Not only the unsubstituted acenes themselves, but also various acene-based molecules with functional side groups and/or heterocycles have been used for prototype and practical organic semiconductor devices. The most recognized acene in the organic electronics field is the five-membered one, pentacene, which has been attracting wide attention because of its unique physicochemical properties. A significant charge carrier mobility of 35 cmurements ,5,6.No matter how promising the characteristics of pentacene are, they are not sufficient for realizing practical p–n junction devices such as solar cells because pentacene, and other unsubstituted acenes such as naphthacene (tetracene), are mostly p-type (donor) materials and it is not easy to switch the polarity of these molecules. To make favorable pairs with these p-type aromatic hydrocarbons, a number of complementary n-type (acceptor) compounds have been synthesized by the introduction of elements of higher electronegativities such as halogens and nitrogen to their backbones ,9,10,11.60 molecules form heteroepitaxial overlayers of an in-plane mean crystallite size of over 100 nm on the single-crystal surfaces of pentacene and rubrene [In this study, the structural and electronic properties of a heteroepitaxial molecular p–n heterojunction formed by stacking TANC on PnSC surfaces were investigated by non-contact mode atomic force microscopy (nc-AFM), grazing-incidence X-ray diffraction (GIXD), X-ray and ultraviolet photoelectron spectroscopy (XPS and UPS), and ultraviolet-visible absorption spectroscopy (UV-vis). Heteroepitaxy has been used as a fundamental technology for obtaining highly ordered semiconductor crystallites and can also be applied for van der Waals molecular materials ,14. For  rubrene ,16,17,18 rubrene . The pre−4 Pa) to prepare the samples. The deposition rate of TANC was set at 14–18 pm/s unless otherwise noted.PnSC platelets were prepared using a physical vapor transport technique and were fixed on Au-coated  or bThe crystallographic structure of the TANC/PnSC heterojunctions was determined by GIXD conducted at BL19B2 of SPring-8. The X-ray wavelength and glancing angle were fixed at 1.00 Å and 0.12°, respectively. Two-dimensional GIXD (2D-GIXD) images were collected by using a PILATUS-300K detector. The camera length was fixed at 172.6 mm from the rotation center of the sample azimuthal angle φ that was rotated 360° at the 0.5° interval. The details of the experimental setup are described elsewhere ,18. The sV = −5 V). The excitation energies were calibrated by using Ta4f7/2 peaks (with binding energy (BE) of 21.8 eV [The XPS and UPS experiments were carried out at BL-13B  of the P 21.8 eV ) or the An nc-AFM image of a PnSC sample with 20 nm-thick TANC is shown in zq = 4.45 nm−1 and 8.90 nm−1 correspond to the (001) and (002) diffraction spots, respectively, of the PnSC substrate [zq = 8.45 nm−1 is attributable to the (020) reflection of the reported crystal structure of TANC [b* axis perpendicular to the PnSC surface. From the TANC diffraction peak width, an out-of-plane mean crystallite size is evaluated to be 26 (±3) nm by the Scherrer equation. This value is in good agreement with the island height seen in the AFM image. It is worth noting that the TANC(020) diffraction peak width for a 50 nm-thick TANC/PnSC sample was identical to that of the 20 nm-thick sample, suggesting that the in-plane mean crystallite size was substantially independent of the TANC thickness, at least up to 50 nm-thick.ubstrate , whereasb*-oriented TANC crystal. The surface lattice of PnSC, c-axis of TANC, in which two adjacent TANC molecules make contact side-by-side with dual CH∙∙∙N hydrogen bonds [60 and perfluoropentacene on PnSC [ on PnSC ,19.3 for the unit cell volume for pentacene and TANC, respectively [ectively ,34; and ectively  are usedWide-range UPS spectra of the PnSC and TANC/PnSC interface are shown in 60 (0.75 (±0.25) eV) [60/pentacene heterojunctions [Whereas it is also necessary to know the LUMO positions of both pentacene and TANC to present a complete picture of the interface electronic states, UPS hardly provides any information about the unoccupied electronic states. The HOMO-LUMO gap width of solid-state pentacene has been reported to be 2.2 eV based on reliable UPS and inverse photoelectron spectroscopy results . In orde.25) eV) . This suunctions .b*–orientation and by aligning its c-axis along the 60/PnSC heterojunction [In this study, the structural and electronic properties of a p–n heterojunction of TANC molecules stacked on PnSC substrates were elucidated. 2D-GIXD demonstrated that TANC grew epitaxially in the junction . The conjunction  utilizin


Phytophthora infestans, is one of the most devastating diseases of potato, and was responsible for the death of millions of people during the Irish Potato Famine in the nineteenth century. Phytophthora infestans is a heterothallic oomycete that typically requires two compatible types (mating types), A1 and A2, to complete sexual reproduction . Oospores have critical effects on disease epidemiology because they serve as the primary inoculum in subsequent growing seasons. The sexual reproduction of Phytophthora species is regulated by α hormones. In previous studies, we proved that transformants in which selected histone deacetylase (HDAC) genes are silenced exhibit abnormal hormone production. In the current study, we compared the transcriptomes of HDAC-silenced and wild-type strains to explore the genes regulated by HDAC and the genes involved in sex hormone biosynthesis in Phytophthora species. A total of 14,423 transcripts of unigenes were identified in the wild-type strain, the HDAC family-silenced transformant (HDST), and the HDAC7-silenced transformant (H7ST). After comparing the intergroup gene expression levels, 1,612 unigenes were identified as differentially expressed among these strains. The expression levels of 16 differentially expressed genes (DEGs) were validated by quantitative real-time PCR. The functional annotation of the DEGs by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that HDACs affect the expression of genes related to metabolic and biosynthetic processes, RNA processing, translation, ribosome biogenesis, cellular structural constituents, RNA binding, and protein binding. Moreover, HDAC7 specifically influences the transcription of genes associated with transport, methylation, mitochondria, organelle inner membranes, receptors and transporters, and hydrolase activities. We also identified 18 candidate genes related to α hormones biosynthesis, including a gene encoding the NF-Y transcription factor (PITG_10861). The overexpression of PITG_10861 increased the production of hormone α2. The results of this study revealed P. infestans genes affected by histone acetylation. The data presented herein provide useful inputs for future research on the epigenetic mechanisms and mating behaviors of Phytophthora species.Late blight caused by  Phytophthora species are filamentous organisms that cause severe diseases in many plant species, resulting in substantial economic losses in the global agricultural industry. Phytophthora species are oomycetes, which form a specific eukaryotic group in the kingdom Stramenopila, and are phylogenetically close to photosynthetic algae  producing specific hormones to initiate selfing and hybridizations in cocultures of the same species  to regulate the extent of the H3K9 acetylation in the ethylene signaling pathway and mediate transcriptional repression  P. infestans MX5-1 strain and two HDAC-silenced transformants for RNA sequencing (RNA-seq) and comparative transcriptome analyses. We then screened for genes related to hormone biosynthesis by comparing the gene expression profiles with the phenotypes of the strains.We previously analyzed the expression of l stages . We alsoHDAC family-silenced transformant HDST43 and the HDAC7-silenced transformant H7ST20 were analyzed in this study  sequence that is conserved in P. infestans HDAC genes. The HDAC7-silenced transformants were generated using vectors containing the 5′ and 3′ untranslated regions of PiHDAC7. The down-regulated HDAC expression of gene-silenced transformants was confirmed by quantitative real-time (qRT)-PCR analyses , and then diluted to 1 ng/μl before checking the insert size with the Agilent 2100 Bioanalyzer  and by quantitative PCR to ensure accuracy. Libraries were sequenced with the Illumina HiSeq X Ten RNA-seq platform . The resulting raw data were transformed into sequenced reads by base calling. The raw data were recorded in a FASTQ file, which contained sequence information (reads) and the corresponding sequencing quality details. Each biological replicate was treated as an independent sample. Raw reads were then filtered to remove reads containing adapters or those of low quality. All remaining clean reads were mapped to the P. infestans reference genome (the EMBL/Genbank/DDBJ databases under the accession GCA_000142945.1) with the TopHat2 software1  beads and fragmented randomly in fragmentation buffer before first-strand cDNA was synthesized with random hexamers and reverse transcriptase. A custom second-strand synthesis buffer  was added with dNTPs, RNase H, and oftware1 .p-values were adjusted based on q-values. A q-value < 0.05 was set as the threshold for determining significant differences in expression. The DEGs were functionally annotated based on gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. The GO enrichment analysis was completed according to the GO-seq R package-based Wallenius non-central hypergeometric distribution  was considered for examining the sequencing depth and gene fragment lengths . The HTSribution . The manribution .PiEF1) as an internal control. Regarding the cDNA synthesis, 1 μg total RNA was reverse transcribed with the oligo(dT)18 primer and Reverse Transcriptase M-MLV  following the manufacturer’s instructions  and HCl7-7-2 (A2SF) were used as the receptors of the transformant-produced hormones α2 and α1, respectively. To evaluate the hormone production of the transformants, a culture block of the hormone receptor strain (1-day-old) was placed at the center of a Petri plate (60 mm in diameter) and covered with a polycarbonate membrane . A transformant block (4-day-old) was placed on top. The cultures were then incubated in a humid chamber in darkness for 3 weeks. For each test, the oospores produced on each block were counted. All experiments were repeated with three replicates per treatment.The bioassay method developed by Phytophthora species mating types are determined based on the type of hormone production and reception. The silencing of HDAC genes resulted in transformants that differed from the WT strain regarding hormone production. The HDAC family-silenced transformant HDST43 was A2SF (self-fertile and produced more oospores with the A1 strain than with the A2 strain), whereas the HDAC7-silenced transformant H7ST20 was A1SF (self-fertile and produced more oospores with the A2 strain than with the A1 strain). The two transformants produced both hormones α1 and α2. However, the bioassay data revealed that HDST43 produced more hormone α2, whereas H7ST20 produced more hormone α1  were generated for each mycelial sample of these strains. Detailed sequencing results are summarized in To investigate the effect of histone acetylation on gene expression as well as the molecular mechanism underlying the biosynthesis of sex hormones, we conducted next-generation sequencing of WT, HDST, and H7ST P. infestans reference genome. After eliminating the redundant transcripts, 72.68–79.13% of the clean reads for these samples were uniquely mapped to the reference genome (r > 0.98) (The transcriptome assemblies obtained for three strains were pooled and used to assemble full-length transcripts with the e genome . Furthere genome . The trae genome . In thise genome . For eac > 0.98) , suggestP. infestans, we compared the three transcriptomes. We identified 7,142 DEGs between the HDST and WT strains, of which 3,446 and 3,696 DEGs exhibited up- and down-regulated expression, respectively. We also identified 6,900 DEGs between the H7ST and WT strains, including 3,441 and 3,459 DEGs with up- and down-regulated expression, respectively. Additionally, 7,001 DEGs were detected between the H7ST and HDST strains, of which 3,558 and 3,443 DEGs exhibited up- and down-regulated expression, respectively  and expressed in at least one sample (FPKM > 1) for a qRT-PCR assay. The qRT-PCR data were highly consistent with the RNA-seq results . The other five genes , whose expression levels were up-regulated in the HDST and H7ST strains  were consistent with the hormone production levels in the WT, HDST, and H7ST strains  , a homeodomain family gene (PITG_08175), and a CBF/NF-Y family gene (PITG_10861) , suggesting these genes may differentially regulate hormone production.Transcription factors are important regulators of gene expression. A thorough analysis of the families . Our DEGG_10861) . The PITPITG_10861) encoding an NF-Y transcription factor, which contains a CBF/NF-Y/archaeal histone domain, was significantly up-regulated in the HDST and H7ST strains  relative to the corresponding expression levels in the WT and empty vector controls   . In A. thromatin . Additiorulation . The AaMthaliana . We are PITG_07424 and PITG_14018 might be involved in sex hormone biosynthesis. The PITG_07424 expression level was significantly up-regulated in the HDST and H7ST strains, suggesting it may participate in the production of hormone α2. However, PITG_14018 expression was significantly down-regulated in the HDST and H7ST strains, indicating that it may be involved in the production of hormone α1. In A. thaliana, CYP77A6 is an in-chain hydroxylase that functions after CYP86A4, the fatty acid ω-hydroxylase, during the synthesis of 10,16-dihydroxypalmitate, which is required for the synthesis of the cutin polyester at floral surfaces  may modify the C4 site to form an α-methyl-branching ketone via a dehydrogenation after the addition of an OH group by P450s. The PITG_14018 expression profile suggested that this gene might participate in the oxidation during the biosynthesis of α-methyl-branching ketone on sex hormone α1. The genetic verification of the effects of these two cytochrome P450s on α hormone production and their roles in α hormone biosynthesis is in progress.surfaces . Althoug50 genes . ConsequPhytophthora species. Previous studies proved that MAV and MEP pathways are mainly involved in the production of the terpenoid backbone (Phytophthora species. This suggests that only the MAV pathway exists in these oomycetes. After the terpenoid backbone is formed, diterpene compounds can be formed via various pathways, including the pathway involving geranylgeraniol, which is structurally similar to the sex hormones of Phytophthora species. However, orthologs of the genes in these pathways have not been detected in Phytophthora species. Earlier research indicated that monoterpenes, diterpenes, and tetraterpenes, which are synthesized in the plastids of various organisms, are derived from the isopentenyl diphosphate produced by the MEP pathway (P. infestans. However, their roles in α hormone synthesis remain to be investigated.Considering the diterpene structures of α hormones, we also screened for genes related to terpene synthesis in backbone . However pathway . However pathway . The ambP. infestans genes affected by histone acetylation, including genes related to the biosynthesis of sex hormones and the associated regulation. The data presented herein provide useful inputs for future investigations on the epigenetic mechanisms and the regulation of mating behaviors in Phytophthora species.This study revealed https://www.ncbi.nlm.nih.gov/bioproject/PRJNA 608542.All RNA-Seq data generated in this study can be found in PRJNA608542, L-YG and X-WW conceived and designed the experiments and wrote the manuscript. X-WW, J-LL, and Y-RS performed the experiments. X-WW and J-LL analyzed the data. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients' viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then,  Betacoronavirus genus. Multiple sequence alignments revealed that SARS-CoV-2 is closely related to bat-derived SARS-like coronaviruses (89% similarity), but when compared with severe acute respiratory syndrome coronavirus (SARS-CoV) and middle east respiratory syndrome (MERS-CoV), SARS-CoV-2 showed less genetic similarity  [In late December 2019, some patients affected by viral pneumonia were found to be epidemiologically associated with the Huanan seafood market in Wuhan, China , 4. It ictively) . In somectively) . A variaN = 500), which eliminates the need for the RNA extraction step. The aim of the current study was, then, to validate an in-house method for isolating viral RNA from nasopharyngeal swabs to be tested by quantitative reverse transcriptase quantitative PCR (RT-qPCR).Viral RNA detection is the main, fastest, and most sensitive test for the diagnosis of SARS-CoV-2 infection . An apprμL of a 30 mg/mL 5 (Merck KGaA) solution in Hanks' Balanced Salt Solution (HBSS) w/calcium and magnesium (Merck) was added to 100 μL of UTM® tube-derived medium. The plate was heated for 15 min at 55°C, denatured for 5 min at 98°C, and then placed for 2 min at 4°C.For SARS-CoV-2 RNA detection, samples from 30 positive and 30 negative subjects were collected with UTM® tubes (COPAN Diagnostics Inc.). Ethical approval was obtained from the Medical Research Ethics Committee of the Region Friuli Venezia Giulia, Italy (CEUR-2020-Os-033). In a 96-well plate, 10 μL of medium from 3 mL of UTM® tube was used as the template, and samples were eluted in 100 μL elution buffer.As a gold standard method for viral NA extraction, the ELITe InGenius® SP200 System (ELITech Group SAS) was used, following the manufacturer's instructions: 200 μL Roche Master, 0.5 μL Reagent mix (containing primers and probes for the envelope (E) gene according to the German Consiliary Laboratory for Coronaviruses ) [μL RT enzyme, 5 μL sample/positive control, and nuclease-free water were mixed to a total volume of 15 μL. The human RNase P gene primer and probe set were used as an internal positive control to monitor sample quality, and RNA isolation. RT-qPCR was performed by the LightCycler® 480 II Instrument (Roche), and absolute quantification was assessed by the LightCycler® 480 II System (Roche).To detect viral RNA, LightMix® Modular SARS and Wuhan CoV E-gene (Roche) were used. Briefly, 4 Berlin)) , 0.1 μL E gene in a given sample that can be clearly distinguished from the background with 95% probability, ensuring a false positive rate ≤ 5%. The Grubb's test was used to identify the outliers, and 46 data points were used for the analysis. The distribution of data was checked with the Shapiro-Wilk test.The LoD was assessed by 5 serial dilutions (5-fold each) of viral RNA isolated from a positive patient with the in-house method and assessed by RT-qPCR. Each standard point was analyzed in 10 replicates. In our case, LoD represents the lowest amount of copies of the To assess sensitivity and specificity of the in-house method, a total of 60 samples, divided in 30 SARS-CoV-2-positive and 30-negative samples, were used.Bland-Altman analysis was used to evaluate the agreement between our in-house method and the gold standard (Elite InGenius SP200 System-mediated automated RNA extraction) . MoreoveThe linear range of the in-house method was established with 7 serial dilutions of viral RNA assessed by RT-qPCR. Each dilution was tested 4 times. The relationship between the observed values and the one obtained with the gold standard was examined with the linear regression. The efficiency was calculated based on the slope of the dilution series.The precision was estimated by performing the RT-qPCR of the same sample under specific conditions. Repeatability was assessed by testing a positive sample 3 times on the same day at 3 different concentrations. Reproducibility (intermediate precision) was evaluated by analyzing the same sample 3 times each day for 3 consecutive days at different concentrations. The Shapiro-Wilk test was used to check the distribution of each series. Levene's test was used to assess the equality of variances in repeatability test and intermediate precision was assessed by one-way ANOVA according to ISO 5725 guidelines.p value ≤ 0.05 was considered statistically significant .All statistical analyses were performed with GraphPad Prism 6.0. A N = 30 positive and N = 30 negative using the gold standard method). N = 30) samples showed no amplification profile. RT-qPCR was performed, and after filtering data with the aforementioned threshold value, 90% sensibility and 100% specificity were found . To further assess agreement, Cohen's kappa was calculated. This is a statistical coefficient representing the degree of accuracy, ranging from 0.01 to 1.00. Referring to data in The result of LoD assessment is shown in re found . The Blare found . The SpeThe extent of linearity of our method was determined by serial dilutions of a SARS-CoV-2-positive specimen . The results of linear regression are summarized in p value calculated with Levene's test was p = 0.17 indicating that there was no significant difference between the measurements. Intermediate precision was evaluated with the same set of dilutions reproduced for 3 consecutive days. The results are shown in Repeatability was evaluated by testing 3 dilutions of a SARS-CoV-2-positive sample 3 times daily. The Following the declaration of pandemic in early 2020, national and local public health laboratories have addressed the crucial problem of detecting the newly isolated pathogen , in ordeE gene, indicated as the most sensitive combination of Roche's modular LightMix® kit. This is a key step as identifying infected subjects immediately triggers containment strategies to reduce the chance of infection. Among the most important performance parameters for a diagnostic procedure are those related to the minimum amount of target that can be detected [p = 0.17).Here, we describe a rapid and cost-effective method that can be applied to analyze swab-derived viral RNA. The strength of this method is its ability to detect as low as 10 copies of the SARS-CoV-2 NA with high sensitivity and specificity . An obvious advantage compared to the gold standard  is certainly that this in-house method is faster, allowing to process 96 samples in about 20 minutes halving the processing time of the samples. Furthermore, by avoiding RNA extraction, the cost per sample is drastically reduced. These features make it useful for rapid sample screening, as in a pandemic outbreak. To validate this method, we have used primers and assays with probes targeted to the detected . Our in-E gene assay. This is a rapid workflow that can help the healthcare system in the prompt identification of infected patients, as is necessary during a pathogen-related outbreak.In summary, we have developed a SARS-CoV-2 extraction-free isolation method suitable for RT-qPCR detection with the


Hypoxia-induced decline in exercise capacity is ubiquitous among lowlanders who immigrated to high altitudes, which severely reduces their work efficiency and quality of life. Although studies have revealed that hypoxia-induced cardiovascular dysfunction limits exercise capacity at high altitudes, the mechanisms have not been well explored at the molecular level. miR-199a-5p is hypoxia-sensitive and serves as an important regulator in cardiovascular pathophysiology. However, whether miR-199a-5p is involved in cardiovascular dysfunction at high altitudes and contributes to subsequent reductions in exercise capacity remains unknown. Thus, this study aimed at exploring these relationships in a high altitude population.A total of 175 lowlanders who had immigrated to an altitude of 3,800 m 2 years previously participated in the present study. The level of plasma miR-199a-5p and the concentration of serum myocardial enzymes were detected by qRT-PCR and ELISA, respectively. Indices of cardiovascular function were examined by echocardiography. The exercise capacity was evaluated by Cooper’s 12-min run test and the Harvard Step Test. Furthermore, we explored the biological functions of miR-199a-5p with silico analysis and a biochemical test.p < 0.001). miR-199a-5p accurately identified individuals with poor exercise capacity . The level of miR-199a-5p was positively correlated with cardiovascular dysfunction indices . Furthermore, miR-199a-5p was involved in the oxidative stress process.The level of miR-199a-5p was significantly higher in individuals with poor exercise capacity at 3,800 m, compared with those with good exercise capacity (In this study, we reported for the first time that the level of circulating miR-199a-5p was positively associated with exercise capacity during chronic hypoxia at high altitudes. Moreover, higher miR-199a-5p was involved in hypoxia-induced cardiovascular dysfunctions, thus contributing to poorer exercise endurance at high altitudes. With the rapid development of the economy, millions of lowlanders (such as the Chinese Han) have immigrated to high altitude regions  for work, construction, and military operations. During exposure to high altitude regions, hypoxia-induced decline in exercise capacity is ubiquitous among them, which severely reduces their work efficiency and quality of life . Subsequvia degrading or blocking mRNA translation. Thus, they play an important role in various physiological and pathophysiological processes, including cardiovascular functions (p < 0.01) after entering high altitude  and 78 kDa glucose-regulated protein (GRP78) (MicroRNAs (miRNAs), which are small non-coding RNA molecules, regulate gene expression post-transcriptionally unctions . Using aaltitude , which ialtitude , only mialtitude . Emerginaltitude . More imet genes . Recentl (GRP78) . HoweverTherefore, in the present study, we examined the expression level of miR-199a-5p in plasma of 175 Han Chinese who had immigrated to high altitude regions 2 years previously. Moreover, we compared the expression level of miR-199a-5p between participants with good and poor exercise capacity at high altitude. Furthermore, we analyzed the association between miR-199a-5p, indices of cardiovascular function, and parameters of exercise capacity. Based on these observations and analyses, we speculate that the level of circulating miR-199a-5p can serve as an important indicator for cardiovascular fitness and exercise capacity during chronic high altitude exposure.In total, 175 young healthy male lowlanders who had immigrated from sea level to high altitude regions  2 years previously were enrolled. They were all emergency rescue personnel with relatively good physical fitness due to regular training. All the participants were ethnic Chinese Han and aged between 18 and 27 years old. The protocol was approved by the Ethics Committee of Army Medical University and performed in accordance with the requirements of the Declaration of Helsinki. All participants provided written informed consent before they were enrolled.All the procedures were processed at 3,800 m with the help of medical staff at the local hospital. A day before the evaluation of aerobic exercise capacity (9:00 a.m.), demographic data and blood samples were collected. Then, the echocardiographic examination and physiological measurement were performed for all the participants. To evaluate the aerobic exercise capacity (9:00 a.m.), all participants carried out the exercise capacity test (Cooper’s 12-min run test). Then, after a six-hour rest, they next took a Harvard step test to assess cardiorespiratory fitness. During the investigation, medical monitoring and emergency first aid treatment were all available. The trail flow diagram of this study is shown in 2), heart rate (HR), diastolic blood pressure (DBP), and systolic blood pressure (SBP), were measured by physicians using a TuffSat Handheld Pulse Oximeter  and sphygmomanometer . Then, after taking a 30-min rest, the echocardiographic indexes of the participants were recorded by sonographers using the color Doppler ultrasound system equipped with a 2.5 MHz adult transducer , including cardiac output (CO), left ventricular ejection fraction (LVEF%), mitral E A peak flow velocity ratio (ME/A), the Tei index for left ventricular function (LV-Tei), mean pulmonary artery pressure (mPAP), and the Tei index of the right ventricle (RV-Tei). Sonographers of the local hospital performed all the measurements and calculations of the echocardiographic indexes.A self-report questionnaire collected demographic data. The basic physiological parameters including pulse oxygen saturation . Finally, all the plasma and serum were transferred into RNase-free tubes and stored at −80°C.At 9:00 a.m., fasting venous blood samples were collected from participants with EDTA tubes and pro-coagulation tubes, respectively. Then, plasma and serum were separated by centrifugation at room temperature (speed: 3000 2max), we employed Cooper’s equation [VO2max (mL/kg min) = 22.351 min × 12 min running distances (km) – 11.288]  . On the contrary, if the VO2max value was under 43 ml/kg min, the participant was considered to have relatively poor exercise capacity (PEC).Participants underwent a 12-min run test on a flat and straight road. They were highly encouraged to run as fast as possible. Then, the finishing point of each participant was marked, and the total running distance in 12 min was calculated. To estimate the value of maximal oxygen uptake (VO 11.288] . For thef China)  because 2 before stepping, at the end of stepping, and during the 5-min recovery were detected by the TuffSat Handheld Pulse Oximeter .The Harvard step test was used to assess cardiorespiratory fitness at a fixed load . ParticiC. elegans miR-39 miRNA mimic)  was added as an endogenous control. Then, the total miRNA was extracted with the miRNeasy Serum/Plasma Kit  according to the manufacturer’s instructions. Next, 6 μL of total miRNA was subjected to further reverse transcription and quantitative real-time PCR (qRT-PCR) reaction by commercial kits  and the CFX Connect Real-Time PCR Detection System . The calculation of the relative expression of miR-199a-5p was performed by the 2CT–Δ method.miRNA was purified from the 200 μL plasma samples. At first, 3.5 μL of work solution of the miRNeasy Serum/Plasma Spike-In Control (The concentration of serum cardiac-specific troponin I (cTnI) was measured by the Cardiac Troponin Assay Kit  based on the instructions of the manufacturer. To analyze the concentration of the serum MB isoenzymes of creatine kinase, the Human CK-MB ELISA Kit  was used following the manufacturer’s protocols.p-value threshold (0.05).MicroT-CDS v5.0 and TarBase v7.0 were employed to predict the target genes of miR-199a-5p . Then, tThe plasma concentration of markers associated with oxidative stress  were determined with the Lipid Peroxidation MDA Assay Kit , Total Superoxide Dismutase Assay Kit , and Human HO-1 ELISA Kit  according to the manufacturer’s instructions.t-test was used, and if not, a Mann–Whitney U test was used. Cohen’s d value and r value [r = z/(sqrt N)] were determined as the effect size measures, respectively. Receiver operating characteristic (ROC) curves were calculated to evaluate the power of miR-199a-5p to distinguish PEC from the GEC groups. Spearman’s correlation was applied to analyze the relationship between variables, and the parameter of effect size was denoted by r. Statistical analyses were performed with IBM SPSS Statistics 19 . P-value ≤ 0.05 was considered statistically significant.The Shapiro–Wilk test was utilized for checking the normality of variables. Then, the normally distributed variables were presented as mean ± standard deviation, and the median (interquartile range) was employed to exhibit non-normally distributed variables. In order to assess the difference of variables, if the variables were normally distributed between the GEC and PEC groups, the independent p < 0.001, Cohen’s d = 2.181), and had a lower VO2max [39.62 ± 3.70 mL/(kg min) vs. 47.61 ± 3.53 mL/(kg min), p < 0.001, Cohen’s d = 2.210, For the distribution of participants with different grades of exercise capacity at 3,800 m, there were 113 participants in the GEC group, and the number of participants with PEC was 62. Compared with the GEC group, the PEC group performed worse in the 12-min running test , and RV-Tei  compared with the GEC group, but no difference was found in left ventricular systolic or diastolic function . For the value of serum myocardial enzymes, the PEC group had higher values of cTnI , and CK-MB , compared with the GEC group.Regarding demographic and basic physiological variables, there was no significant difference between the two groups. For the echocardiographic indexes, the PEC group had a higher value of mPAP  and serious myocardial injury (higher cTnI and CK-MB) related to higher miR-199a-5p might have contributed to the decline of exercise capacity at high altitude.To the best of our knowledge, this study described for the first time a link between circulating miR-199a-5p level and the aerobic exercise capacity of individuals during chronic exposure to high altitude. Moreover, our results also demonstrated that circulating miR-199a-5p was a novel biomarker for identifying individuals with PEC from those with GEC. It is of great significance to objectively evaluate aerobic exercise capacity while people are engaged in mountaineering, sports competition, commercial activity, and military operations at high altitudes, in order to decrease accidents and improve their work performance. Because it is difficult and dangerous to measure maximum exercise capacity by various exercise tests in a high altitude hypoxic condition, the index from body fluid reflecting cardiopulmonary endurance can be used as an alternative method with acceptable accuracy. Circulating miRNAs are considered specific, stable, and easily detectable biomarkers and have been applied successfully in diagnosing various diseases . In the via agents could alleviate pulmonary hypertension and myocardial injury, thus improving cardiovascular function  Ethics Committee, China. The patients/participants provided their written informed consent to participate in this study.PL and YG conceived and designed the study. BX oversaw laboratory analyses. PL provided the overall supervision of the study. HH, ZZ, and PH completed the statistical analysis or contributed to the laboratory experiments. SX and XG contributed to sample and data collection. HH drafted the report. PL and YG were the guarantors. All authors contributed to the interpretation of the results, critical revision of the manuscript, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Study of the three genes of this family, mcwc-1a, mcwc-1b, and mcwc-1c, resulted in a marked functional specialization, as only mcwc-1a was essential to maintain the virulence potential of M. lusitanicus. The traditional role of wc-1 genes regulating light-dependent responses is a thoroughly studied field, whereas their role in virulence remains uncharacterized. In this work, we investigated the mechanism involving mcwc-1a in virulence from an integrated transcriptomic and functional approach during the host–pathogen interaction. Our results revealed mcwc-1a as a master regulator controlling an extensive gene network. Further dissection of this gene network clustering its components by type of regulation and functional criteria disclosed a multifunctional mechanism depending on diverse pathways. In the absence of phagocytic cells, mcwc-1a controlled pathways related to cell motility and the cytoskeleton that could be associated with the essential tropism during tissue invasion. After phagocytosis, several oxidative response pathways dependent on mcwc-1a were activated during the germination of M. lusitanicus spores inside phagocytic cells, which is the first stage of the infection. The third relevant group of genes involved in virulence and regulated by mcwc-1a belonged to the “unknown function,” indicating that new and hidden pathways are involved in virulence. The unknown function category is especially pertinent in the study of mucormycosis, as it is highly enriched in specific fungal genes that represent the most promising targets for developing new antifungal compounds. These results unveil a complex multifunctional mechanism used by wc-1 genes to regulate the pathogenic potential in Mucorales that could also apply to other fungal pathogens.Mucolares are an ancient group of fungi encompassing the causal agents for the lethal infection mucormycosis. The high lethality rates, the emerging character of this disease, and the broad antifungal resistance of its causal agents are mucormycosis features that are alarming clinicians and researchers. Thus, the research field around mucormycosis is currently focused on finding specific weaknesses and targets in Mucorales for developing new treatments. In this work, we tested the role of the  The family of wc-1 genes has been associated with virulence in both Ascomycetes and Basidiomycetes [Mucormycosis is a threatening infection caused by Mucorales that is the most unexplored host–pathogen interaction . The recomycetes ,15,16; hwc-1 genes control most of the physiological responses of fungi to light. Mutations in these genes provoke the lack of light-regulated responses like the production of carotenes, the control of the circadian clock, sporulation, and phototropism [wc-1 genes regulating different responses to light has been intensely studied and dissected in several fungi since they were firstly described in Neurospora crassa [Fusarium oxysporum, Cryptococcus neoformans, Cercospora zeae-maydisa, and Botrytis cinerea [wc-1 genes in virulence is the same in different fungi, with these genes being necessary to maintain full virulence. In addition, wc-1 genes regulate virulence in both plant- and animal-specific fungal pathogens. In F. oxysporum, which infects both plant and animal hosts, lack of wc-1 gene only affected virulence in animal hosts [C. neoformans have suggested that the role of wc-1 genes in virulence is independent of light [wc-1 proteins showed that light-sensing impaired mutants were still fully virulent [The otropism ,18,19. Totropism . Thus, totropism . The rola crassa . However cinerea ,15,16,23of light . In thesvirulent .wc-1 genes to virulence. However, several studies of the fungus Mucor lusitanicus showed a detailed dissection of a complex wc-1 genes regulatory system, characterizing all the responses controlled by light and these genes [M. lusitanicus, there are three different orthologous genes, which acquired functional specialization concerning the different responses triggered by light [mcwc-1a, mcwc-1b, and mcwc-1c. First, mutagenesis analysis of these three genes showed that mutants in mcwc-1c were defective in the carotene production, whereas mcwc-1a mutants lacked the phototropism response [mcwc-1b is involved in regulating carotene production and vegetative growth, but in a more complex pathway, including the negative regulator gene crgA [In Mucorales, there are no studies relating the se genes . In M. lby light . These tresponse . A seconene crgA ,26,27,28wc-1 genes in the virulence of Mucorales, finding that indeed virulence is also dependent on this family of genes. As expected from the functional specialization of the three mcwc-1 genes in the responses triggered by light, virulence is specifically controlled by only one of the three genes, mcwc-1a. After considering these results, we analyzed the pathways regulated by wc-1 genes related to virulence, which have not been explored in any other fungi. Our group previously developed a system to study host–pathogen interactions using fungal spores phagocyted by macrophages [mcwc-1a during phagocytosis, finding an intricate gene network involved in the process.Here, we explored the role of the rophages . In thisM. lusitanicus strain CBS277.49 served as the parental strain of all the following strains. The mutants in white collar-1 genes (mcwc-1a∆ (MU242), mcwc-1b∆ (MU244), and mcwc-1c∆ (MU247)) were generated in a previous study [wc-1 mutants, except for the aforementioned deletions. M. lusitanicus strain NRRL3631 is an independent natural isolate that was used as an avirulent mock control in the virulence assays [wc-1a∆ MU2, mcwc-1us study  because e assays .® 70 µm cell strainers to remove unintentionally harvested mycelium pieces before confronting macrophages or animal models.Fungal colonies were grown in a solid, rich YPG medium with pH adjusted at 4.5 and an incubation temperature of 26 °C for optimal growth and sporulation . Spores 6 spores [mcwc-1a (MU242 and MU243). During this procedure, the mice were anesthetized using isoflurane via inhalation and monitored until they recovered from the anesthesia. Mice were housed under established conditions with free food and autoclaved water. The animal welfare was checked twice a day for 20 days, and those mice following the criteria for discomfort were euthanized by CO2 inhalation. Survival rates during the time were plotted in a Kaplan–Meier curve (Graph Pad Prism), and differences were considered statistically significant with a p-value ≤ 0.05 in a Mantel–Cox test.Male OF-1 mice weighing 30 g and two months old  ,33 were 6 spores . The assmcwc-1a∆ and the wild type R7B were confronted with a mouse macrophage cell-line (J774A.1 or ATCC TIB-67) in a 1.5:1 ratio as described in previous studies to perform host–pathogen interaction assays [Spores from mutant strain n assays . BrieflyDuplicates of each sample were pooled together, and RNA was extracted using the standard procedure of the RNeasy plant minikit , resulting in two replicated RNA extractions per sample that were sent for sequencing to BaseClear . There, mRNA was enriched by poly(A) purification capture, and cDNA libraries were prepared using TruSeq RNA library preparation kits. The cDNA library was sequenced using an Illumina HiSeq 2500 system, producing two replicated raw datasets of first-stranded (or reverse), 50 bp, single-end reads.M. lusitanicus wild-type and mcwc-1a∆ single- and co-cultures with mouse macrophages was assessed with FASTQC v0.11.8 before and after removing adapter and contaminant sequences using Trim Galore! v.0.6.2 (available at http://www.bioinformatics.babraham.ac.uk/projects/ (accessed on 19 February 2021)). Reads with low Phred quality score (Q ≤ 32) and/or total length (length ≤ 20 nt) were filtered out, as well as adapter sequences with an overlap longer than ≥4 bases in the 3′ end of the read. These high-quality and adapter-free reads were mapped to the M. lusitanicus f. lusitanicus v2.0 genome (herein Mucci2 [https://mycocosm.jgi.doe.gov/Mucci2/Mucci2.info.html (accessed on 19 February 2021)) using STAR v.2.7.1a [limma package v.3.38.3 [2 FC) ≥ 1.0, and average count per million reads (CPM) in log2 units ≥ 0.0). Average gene expression values (as log2 CPM) and log2 FC ratios were correlated among samples with scatter plots, highlighting up- and down-regulated genes and three housekeeping genes whose expression is equal among samples: translation elongation factor EF-1 (Mucci2 ID: 156959), RNA polymerase III transcription factor IIIC subunit TFIIIC (Mucci2 ID: 106349), and vacuolar-type H+-ATPase V-ATPase (Mucci2 ID: 154376).The quality of the raw reads from n Mucci2 , availabv.2.7.1a . Read cov.2.7.1a  excludinv.3.38.3 , using rv.3.38.3 : false dhttps://cran.r-project.org/web/packages/pheatmap/ (accessed on 19 February 2021)). Eukaryotic Orthologous Groups (KOG) and Gene Ontology (GO) terms were identified with EggNOG-mapper v2.0 [p-value ≤ 0.05 in a one-sided Fisher’s exact test. GO term enrichment was also analyzed using FungiDB (available at https://fungidb.org/fungidb/app (accessed on 19 February 2021)), plotting the enriched and non-redundant GO terms with REVIGO (available at http://revigo.irb.hr/ (accessed on 19 February 2021)).DEGs were clustered with Canberra distance and Ward’s clustering method to plot a heatmap using the pheatmap package v1.0.12 . In contrast, lacking any of the other two wc-1 genes, mcwc-1b or mcwc-1c, did not alter the virulence potential of M. lusitanicus because the corresponding mutants showed a similar pathogenic behavior as the virulent wild-type R7B strain . These results indicated that in the M. lusitanicus wc-1 gene family, the gene mcwc-1a has a specialized role in virulence, although a partial redundancy of the other two genes cannot be excluded.The mcwc-1a in the virulence of Mucorales and its consequent functional conservation throughout the fungal kingdom, we explored the transcriptional response controlled by mcwc-1a both during regular growth and macrophage phagocytosis, trying to identify the genes required for the fungal response during the first stage of the host–pathogen interaction. We conducted a deep-sequencing approach (RNA-seq) using mRNA from spores of the wild-type R7B strain and the mutant mcwc-1aΔ grown in two different conditions (both in the dark). The first condition mimicked a saprotrophic environment, using the rich medium L15. The second condition was a co-culture of spores and the J774A.1 cell-line of mouse macrophages (1.5:1 spore–macrophage ratio) for 5 h, ensuring the phagocytosis of all the spores. This second condition is a novel study model in Mucorales to investigate the initial step of fungal infections, in which the pathogen must overcome the innate immune response to continuing the infection and tissue invasion [mcwc-1aΔ mutant had significantly different profiles in the two growth conditions. The analysis showed that a reduced proportion of the genes controlled by mcwc-1a are involved in response to phagocytosis, the first step of the fungal infection  ≤ 0.05) and an absolute log2 fold change (FC) greater than or equal to 1 (log2 FC ≥ |1.0|). Thus, a total of 3456 genes (mcwc-1a (1957 upregulated and 1499 downregulated) during saprotrophic conditions. In contrast, after phagocytosis, only 478 genes showed differential expression controlled by this gene (172 upregulated and 306 downregulated)  . There amcwc-1a were identified, we performed an enrichment analysis of Eukaryotic Orthologous Groups (KOG), exploring the putative cellular processes controlled by this gene both under saprotrophic conditions (rich medium) and macrophage phagocytosis  without affecting virulence. However, these results do not discard the regulation of mcwc-1a of other unknown secondary metabolites with a putative role in virulence. Our results showing the functional specialization of the three wc-1 genes in M. lusitanicus could suggest another hypothesis for its role in virulence. The gene related to virulence, mcwc-1a, also controls phototropism. Tropism is an essential process for growth and sporulation and also could be related to the pathogenic potential because it might control the hyphae movement during tissue and blood vessel invasion for dissemination. Thus, a set of genes involved in tropism and controlled by mcwc-1a could link to virulence.Once the conservation of the essential role of WC proteins in virulence has been established in Mucorales, the next question to resolve relates to the mechanism used by these proteins to regulate virulence. In 1 mutant . Other sirulence . In M. lmcwc-1a showed a substantial number of genes  differentially expressed during saprotrophic growth conditions. The general interpretation of these results clearly defines the wc-1 genes as master regulators controlling the development of M. lusitanicus. However, a more specific and reduced set of genes was found differentially expressed during the interaction with macrophages, in a context simulating the first stage of the infection. A detailed analysis clustering all the differentially expressed genes and dissecting their putative functional associations led us to suggest three non-exclusive hypotheses to explain the role of WC proteins in controlling virulence. First, we have repeatedly found groups of genes in different clusters involved in the protection against oxidative damage. This process is an essential fungal function required both after the exposition to the light and during the macrophage confrontation, linking the traditional response of WCs with its role in controlling virulence. Thus, our results suggest that mcwc-1a might be influencing the oxidative-damage protection pathways in the two scenarios. Secondly, we found an enrichment of differentially expressed genes with unknown functions during the interaction with the macrophages. These results indicate that besides the protection against oxidative stress, mcwc-1a must be controlling other cellular processes with essential roles in the survival inside the macrophages. This group of unknown function genes in Mucorales contains a large proportion of unique genes not found in other organisms [mcwc-1a in the second stage of the infection, during the tissue invasion, angioinvasion, and dissemination of the infection. This stage could link to essential processes related to tropism, cell motility, and cytoskeleton-reorganization functions. These could be the first results suggesting a link between the regulation of phototropism and virulence by Mcwc-1a in a light-independent manner.Independently of how the WC-dependent mechanism works in regulating virulence, all the studies conclude that finding the WC-dependent genes is the necessary next step to understand this mechanism. Our results investigating the gene network controlled by rganisms , which mmcwc-1a described in this work highlights the oxidative stress, tropism, and a group of unknown functions as the main processes that this master regulator might control to regulate virulence potential in fungal pathogens.The virulence potential of fungal pathogens depends on complex mechanisms controlled by diverse pathways and cellular processes. The intricate gene network controlled by